CYSTIC FIBROSIS is the most common lethal autosomal

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1 Vol. 333 No. 2 EVIDENCE OF A CYSTIC FIBROSIS MUTATION ASSOCIATED WITH MILD LUNG DISEASE 95 A CYSTIC FIBROSIS MUTATION ASSOCIATED WITH MILD LUNG DISEASE KING-HAN GAN, M.D., HENK J. VEEZE, M.D., ANS M.W. VAN DEN OUWELAND, PH.D., DICKY J.J. HALLEY, PH.D., HANS SCHEFFER, PH.D., ANNEMIEKE VAN DER HOUT, PH.D., SHELLEY E. OVERBEEK, M.D., JOHAN C. DE JONGSTE, M.D., PH.D., WILLEM BAKKER, M.D., PH.D., AND HARRY G.M. HEIJERMAN, M.D., PH.D. CYSTIC FIBROSIS is the most common lethal autosomal recessive disorder among whites, with an incidence of about 1 in 25 live births. One person in 25 is an asymptomatic carrier. 1 The gene containing mutations responsible for cystic fibrosis was cloned in It codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel dependent on cyclic AMP (camp). In cystic fibrosis, defective chloride transport across membranes causes a lack of water in external secretions. This leads to tenacious mucus in the lungs and protein plugs in the pancreas and to the characteristically high sweat chloride levels. The cardinal features of the disease are chronic pulmonary infection and exocrine pancreatic insufficiency. The median survival in the Netherlands is currently 27 years (Dutch Cystic Fibrosis Registry: unpublished data), with most patients dying of pulmonary complications. Since the gene for cystic fibrosis was cloned, there have been several studies on associations between the genotype and the phenotype in cystic fibrosis. 5-8 A number of mutations (R117H, R334W, R347P, A455E, and P574H) appear to be associated with pancreatic sufficiency 9 and residual transmembrane transport of From the Adult Cystic Fibrosis Center, Department of Pulmonology, Leyenburg Hospital, The Hague (K.-H.G., W.B., H.G.M.H.); the Department of Pediatrics (H.J.V.) and Division of Pediatric Pulmonology (J.C.J.), Sophia Children s Hospital, Rotterdam; the Departments of Clinical Genetics (A.M.W.O., D.J.J.H.) and Pulmonology (S.E.O.), Dijkzigt University Hospital, Rotterdam; and the Department of Medical Genetics, University of Groningen, Groningen (H.S., A.H.) all in the Netherlands. Address reprint requests to Dr. Heijerman at the Adult Cystic Fibrosis Center, Dept. of Pulmonology, Leyenburg Hospital, 275 Leyweg, 2545 CH The Hague, the Netherlands. Abstract Background. Cystic fibrosis is the most common lethal autosomal recessive disorder among whites. Among Dutch patients with cystic fibrosis, F58 is the most common mutation and A455E the second most common mutation of the cystic fibrosis transmembrane conductance regulator gene on chromosome 7. A455E is associated with preserved pancreatic function and residual secretion of chloride across membranes. We investigated whether it is also associated with less severe pulmonary disease in patients with cystic fibrosis. Methods. A total of 33 patients with compound heterozygosity for the A455E mutation were matched according to age and sex with patients who were homozygous for the F58 mutation. The pairs were analyzed with respect to the following outcome variables: age at diagnosis, pulmonary-function values, and the frequency of pseudomonas colonization, pancreatic sufficiency, and diabetes mellitus. Results. Cystic fibrosis was diagnosed at a later age in the patients with the A455E mutation than in the F58 homozygotes (mean age at diagnosis, 15. vs. 3.1 years; P.1). Fewer patients with the A455E mutation had pancreatic insufficiency (21.2 percent vs percent, P.1), and none had diabetes mellitus ( percent vs percent, P.4). Forced expiratory volume in one second (FEV 1 ) and forced vital capacity (FVC) were significantly higher in the patients with the A455E mutation (mean FEV 1, 73.9 percent of the predicted value vs percent of the predicted value; P.2; mean FVC, 88.7 percent of the predicted value vs percent of the predicted value; P.4). Fewer patients with the A455E mutation were colonized with Pseudomonas aeruginosa (33.3 percent vs..6 percent, P.2). Conclusions. A455E is a common mutation causing cystic fibrosis in the Netherlands. Although several mutations are known to be associated with less severe pancreatic disease, our findings demonstrate a correlation between the A455E mutation and mild pulmonary disease. Because mortality in this disease depends primarily on the progression of pulmonary disease, patients with the A455E mutation have a better prognosis than patients who are homozygous for the F58 mutation. (N Engl J Med 1995;333:95-9.) chloride. 1,11 The most common mutation, F58, is associated with pancreatic insufficiency and severe pulmonary disease. 5,6 There is great variation in the severity of lung disease, but until now no mutation associated with mild pulmonary disease has been found. Recently, we noted that a group of Dutch patients with cystic fibrosis who carried the A455E mutation had significantly better lung function than patients homozygous for the F58 mutation. 12 An association between this mutation and exocrine pancreatic sufficiency has already been described. 9,11 In addition, A455E appears to be associated with residual secretion of chloride in electrophysiologic studies of rectal-biopsy specimens. 11 Among our patients the A455E mutation is relatively common and is associated with an older age at diagnosis. 11,13 Because of the older age at diagnosis and the mutation-dependent residual chloride secretion associated with A455E, we hypothesized that the presence of this mutation could result in milder lung disease. To investigate this possibility, we compared clinical data from a group of patients with cystic fibrosis carrying this mutation with patients homozygous for the F58 mutation who were matched according to age and sex. Patients METHODS A total of 125 adult ( 18 years of age) patients with cystic fibrosis from the Leyenburg cystic fibrosis center in The Hague, 18 children from Sophia Children s Hospital in Rotterdam, and 45 adult patients from Dijkzigt University Hospital in Rotterdam were

2 96 THE NEW ENGLAND JOURNAL OF MEDICINE July 13, 1995 screened for CFTR mutations. These patients represent approximately 3 percent of all known patients with cystic fibrosis in the Netherlands. Among the patients screened, 151 were found to be homozygous for the F58 mutation and 39 were found to have compound heterozygosity for the A455E mutation. In the A455E compound heterozygotes, the following mutations were found on the other allele: F58 (27 patients), G A (4 patients), EX (4 patients), G542X (2 patients), R553X (1 patient), and an unknown mutation (1 patient). The patient with the unknown mutation was excluded from further analysis. The other mutations found in A455E heterozygotes are all associated with pancreatic insufficiency and have been classified as severe cystic fibrosis mutations, 8 predicted to produce no functioning CFTR. 14 Therefore, all patients with an A455E allele were analyzed together. One patient, whose genotype was A455E/ F58, has been described elsewhere. 15 This patient died at the age of 71 and could not be matched with a F58 homozygote because of her advanced age. No other data on deceased A455E compound heterozygotes were available, and therefore no data on deceased patients were included in the analysis of matched pairs. DNA analysis was performed at the University of Groningen and at Dijkzigt University Hospital. Analysis for the F58 mutation was carried out by direct polyacrylamide-gel electrophoresis of the CFTR exon 1 product of the polymerase chain reaction 16 or as part of a multiplex amplification refractory mutation system. 17 For the analysis of the A455E mutation, a specific amplification refractory mutation system was developed (Scheffer H, et al.: unpublished data). Demographic Data Demographic data included each patient s date of birth, sex, and race and ethnic origin. For A455E compound heterozygotes, the place of birth was recorded, as were the birthplaces and family names of their parents and grandparents, as far as could be ascertained. Clinical Evaluation General outcome variables included age at diagnosis and height and weight at last clinic visit. Height percentiles for age were calculated with the use of reference values for Dutch children. 18 Weightfor-height percentiles were calculated with the same reference values. The results of the most recent representative pulmonary-function test (i.e., one not performed during an acute pulmonary exacerbation) were considered. Values for forced vital capacity (FVC) and forced expiratory volume in one second (FEV 1 ) were compared with reference values for the European Union, as endorsed by the European Respiratory Society. 19 We considered patients to be colonized with Pseudomonas aeruginosa if a minimum of three consecutive sputum cultures obtained over a period of at least six months grew this microorganism. The patients were considered to have pancreatic sufficiency if fecal fat excretion was normal ( 1 percent) during three-day fat-balance studies or if they had a normal N-benzoyl-L-tyrosyl-p-aminobenzoic acid test, normal fecal chymotrypsin concentrations, or normal serum b- carotene concentrations when not using pancreatic-enzyme supplements. Patients with abnormal test results were considered to have pancreatic insufficiency. Diabetes mellitus was considered to be present when insulin injections were needed to control blood glucose levels. Statistical Analysis Each of the patients with the A455E mutation was matched with the F58 homozygote closest in age, within two years, and of the same sex. The patient who died at the age of 71 and four other A455E compound heterozygotes (4, 43, 53, and 47 years old, with the first three having a genotype of A455E/1717-1G A and the fourth a genotype of A455E/ F58) could not be matched within two years of age with a F58 homozygote and were excluded. The F58 homozygotes who were entered in the matched-pair analysis were compared with the whole group of F58 homozygotes to determine whether their pulmonary function was representative of that of the group as a whole. In the matched-pair analysis, continuous variables were compared by a two-tailed paired t-test and categorical variables Table 1. Characteristics of Pairs of F58 Homozygotes and A455E Compound Heterozygotes Matched According to Sex and Age.* CHARACTERISTIC NO. OF PAIRS F58 HOMOZYGOTES *Plus minus values are means SD. NS denotes not significant. The following genotypes were identified: A455E/ F58 (25 patients), A455E/EX (4), A455E/G542X (2), A455E/R553X (1), and A455E/1717-1G A (1). By two-tailed paired t-test. Four patients were not old enough for pulmonary-function testing. By two-tailed exact binomial test. Weight was adjusted for height. were compared by computing exact binomial probabilities. To show the progression of lung disease, a regression line for FEV 1 was calculated for all patients in both groups according to age. The slopes of the two regression lines were compared with an analysis-of-covariance model. All P values were two-tailed, and probabilities of less than.5 were considered significant. Data were analyzed with the SPSS statistical package. RESULTS A455E COMPOUND HETEROZYGOTES P VALUE Sex no. (%) 33 Male 16 (49) 16 (49) NS Female 17 (51) 17 (51) Age yr 33 Mean NS Range Age at diagnosis yr FEV 1 % of predicted value FVC % of predicted value Pseudomonas colonization 33 (.6) 11 (33.3).2 no. (%) Pancreatic insufficiency (93.9) 7 (21.2).1 no. (%) Diabetes mellitus no. (%) 33 9 (27.3).4 Weight (percentile) NS Height (percentile) Men Women NS All of the patients were white. One patient (a F58 homozygote) was of Polish descent, and all of the others were Dutch. As far as could be determined, the families of the A455E compound heterozygotes were not related, nor did they come from geographically isolated regions. No other connecting feature, such as religion, could be found among these families. At least four A455E heterozygotes were former cigarette smokers, whereas none of the F58 homozygotes had ever smoked. Table 1 summarizes the results of the matched-pair analysis. Twenty-nine of the patients were matched within one year of age (88 percent), and the remaining four patients were matched within two years of age. The patients with the A455E mutation had significantly better lung function than did the F58 homozygotes (mean FEV 1, 73.9 percent of the predicted value vs percent of the predicted value; P.2). The mean FVC was 88.7 percent for A455E compound heterozygotes and 76.3 percent for F58 homozygotes (P.4). Colonization with P. aeruginosa (P.2) and pancreatic insufficiency (P.1) were significantly

3 Vol. 333 No. 2 EVIDENCE OF A CYSTIC FIBROSIS MUTATION ASSOCIATED WITH MILD LUNG DISEASE 97 FEV 1 (% of predicted value) FEV 1 (% of predicted value) A455E Compound Heterozygotes 3 Figure 1. FEV 1 in 34 A455E Compound Heterozygotes and 13 F58 Homozygotes, According to Age. Solid squares represent patients included in the matched-pairs analysis; 4 A455E compound heterozygotes and 21 F58 homozygotes were not included because they were too young for pulmonary-function testing. The regression lines (solid lines) and 95 percent confidence intervals (dashed lines) are indicated. The regression line had a slope of.89 for A455E compound heterozygotes and.178 for F58 homozygotes (P.2). less prevalent in patients with the A455E mutation, and diabetes mellitus was absent in this group (P.4). Weight (adjusted for height) was normal and not significantly different in the two groups. The men with the A455E mutation, but not the women, were significantly taller than matched F58 homozygotes. Figure 1 shows the distribution of FEV 1 (as a percentage of the predicted values) for all patients according to age. The regression line had a slope of.89 for A455E compound heterozygotes and a significantly steeper slope of.178 for F58 homozygotes (P.2). There were no significant differences in lung function between the F58 homozygotes included in the matched-pair analysis and all other F58 homozygotes. DISCUSSION The results of this study show that there is a relation between the presence of the A455E mutation and milder lung disease in patients with cystic fibrosis. Although the A455E mutation is known to be associated with 4 Age (years) 5 DF58 Homozygotes 3 4 Age (years) pancreatic sufficiency, 9 the association between A455E or any other cystic fibrosis mutation and milder lung disease has apparently not been found before. In earlier studies of the association between genotype and phenotype, large variations in the severity of lung disease among patients with cystic fibrosis and the same genotype have been noted. 8,21,22 Patients who are homozygous for the F58 mutation have been shown to have more severe lung disease than F58 heterozygotes with pancreatic sufficiency. 5,6 A number of studies of disease severity in patients with mutations not involving F58 have been conducted, but the results were inconclusive because the numbers of patients were small. To overcome this problem, the Cystic Fibrosis Genotype Phenotype Consortium initiated a large multicenter study that included 798 patients with cystic fibrosis. 8 Of the eight mutations studied, none were associated with mild lung disease (A455E was not included). R117H, a mutation associated with residual transmembrane chloride transport, 1 was also associated with an older age at diagnosis and with pancreatic sufficiency, but not with better lung function. The severity of disease in cystic fibrosis is determined by the mutation resulting in the least severe disease. 9 All the A455E compound heterozygotes included in our study had a mutation on their other allele that was associated with severe disease. Mild symptoms in these patients are therefore most likely associated with the presence of the A455E mutation. By studying patients seen at two collaborating centers in nearby cities, we minimized the influence of differences in therapy and environmental factors. This is in contrast to the study by the Cystic Fibrosis Genotype Phenotype Consortium, 8 which included 48 centers in 15 countries. Table 2. Frequency of F58 and A455E Mutations in Various Countries. STUDY COUNTRY/REGION *Includes 31 A455E compound heterozygotes from the present study. Blacks were studied. Ashkenazi Jews were studied. NO. OF CHROMOSOMES SCREENED F58 A455E Present study The Netherlands Cystic Fibrosis Genetic The Netherlands* Analysis Consortium 33 Lindner et al. 34 Southwestern Germany 2 67 Cuppens et al. 35 Belgium Super and Schwarz 36 Northwestern England Shrimpton et al. 37 Scotland Claustres et al. 38 Southern France Cutting et al. 39 Baltimore Cutting et al., 39 Zielenski Toronto et al. 4 Rozen et al. 31 Quebec Rozen et al. 31 Saguenay Lac St. Jean, Quebec Cutting et al. 39 United States Cutting et al. 39 Israel 94 3 %

4 98 THE NEW ENGLAND JOURNAL OF MEDICINE July 13, 1995 Our results show that the type of mutation has a significant impact on pulmonary function in cystic fibrosis. Because of the relatively high frequency of the A455E mutation among our patients, we were able to include 33 patients, as compared with the 23 patients with the R117H mutation included in the consortium s report. 8 When sufficient numbers of patients are studied, it should be possible to show that other mutations associated with the preservation of pancreatic function and residual transmembrane chloride transport are also associated with milder pulmonary disease. A455E is a missense mutation that leads to a change from alanine to glutamic acid in amino acid residue 455 of the CFTR protein. 27 CFTR is a chloride transporter driven by camp, and the A455E mutation is situated in the first nucleotide-binding fold, two residues removed from the so-called Walker-A motif, the proposed site of interaction with the phosphoryl moiety of the bound camp. 28 The A455E mutation may interfere with the binding of camp to the CFTR protein. The CFTR protein coded for by the F58 mutation is subject to defective intracellular processing and does not reach the cell membrane, but is degraded intracellularly. 29 The CFTR protein containing the A455E mutation may also be subject to this mechanism, but to a lesser degree. 3 Unlike F58 homozygotes, patients with cystic fibrosis who have the A455E mutation have residual transmembrane chloride transport, 11 a point that increases the likelihood that at least some functioning CFTR protein reaches the cell membrane. The A455E mutation was first found in patients from the Saguenay Lac St. Jean region in northern Quebec. 27,31 In this formerly isolated community, the incidence of cystic fibrosis and other inherited diseases is higher than normal. 32 The A455E mutation is seldom found elsewhere in the world (Table 2), 31,33-4 but it is relatively common in the Netherlands, where it is the second most common cystic fibrosis mutation (3. percent of all cystic fibrosis alleles). 33 The two mutations that we studied, F58 and A455E, account for 8.1 percent of the mutations found in Dutch patients with cystic fibrosis. In our study the frequency of A455E was 7. percent, which may be related to the age distribution of our patients. In our patients, the presence of the A455E mutation was associated with the preservation of both endocrine and exocrine pancreatic function, less frequent colonization with P. aeruginosa, and mild lung disease. The regression line for FEV 1 according to age was less steep in A455E compound heterozygotes than in F58 homozygotes, an indication that the progression of pulmonary disease is slower in patients with the A455E mutation. A455E is the first CFTR mutation for which an association with mild lung disease could be found. Because survival in cystic fibrosis is strongly correlated with the progression of pulmonary disease, 41 we expect that patients with cystic fibrosis who have this mutation will have a better prognosis than patients homozygous for F58. REFERENCES 1. Lewis PA. The epidemiology of cystic fibrosis. In: Hodson ME, Geddes DM, eds. Cystic fibrosis. London: Chapman and Hall, 1995: Rommens JM, Iannuzzi MC, Kerem BS, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245: Riordan JR, Rommens JM, Kerem BS, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. 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5 Vol. 333 No. 2 EVIDENCE OF A CYSTIC FIBROSIS MUTATION ASSOCIATED WITH MILD LUNG DISEASE Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC. Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 1993;9: Cheng SH, Gregory RJ, Marshall J, et al. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 199;63: Ostedgaard LS, Sheppard DN, Welsh MJ. Analysis of mutations in the first nucleotide-binding domain of CFTR which are associated with pancreatic sufficiency. Pediatr Pulmonol 1994;Suppl 1:181. abstract. 31. Rozen R, De Braekeleer M, Daigneault J, et al. Cystic fibrosis mutations in French Canadians: three CFTR mutations are relatively frequent in a Quebec population with an elevated incidence of cystic fibrosis. Am J Med Genet 1992;42: Daigneault J, Aubin G, Simard R, De Braekeleer M. Incidence of cystic fibrosis in Saguenay-Lac-St.-Jean (Quebec, Canada). Hum Biol 1992;64: The Cystic Fibrosis Genetic Analysis Consortium. Population variation of common cystic fibrosis mutations. Hum Mutat 1994;4: Lindner M, Wolf A, Moh B, et al. The spectrum of CFTR mutations in south-west German cystic fibrosis patients. Hum Genet 1992;9: Cuppens H, Marynen P, De Boeck C, Cassiman JJ. Detection of 98.5% of the mutations in Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene. Genomics 1993;18: Super M, Schwarz MJ. Mutations of the cystic fibrosis gene locus within the population of the Northwest of England. Eur J Pediatr 1992;151: Shrimpton AE, McIntosh I, Brock DJ. The incidence of different cystic fibrosis mutations in the Scottish population: effects on prenatal diagnosis and genetic counselling. J Med Genet 1991;28: Claustres M, Desgeorges M, Kjellberg P, Tissot C, D le J. Analysis of 3 known cystic fibrosis mutations: 1 mutations account for 27% of nondelta F58 chromosomes in southern France. Hum Genet 1992;9: Cutting GR, Curristin SM, Nash E, et al. Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians. Am J Hum Genet 1992;5: Zielenski J, Markiewicz D, Xia Y, et al. Genotyping of 515 CF patients in Toronto. Pediatr Pulmonol 1992;Suppl 8:244. abstract. 41. Kerem E, Reisman J, Corey M, Canny GJ, Levison H. Prediction of mortality in patients with cystic fibrosis. N Engl J Med 1992;326: IMAGES IN CLINICAL MEDICINE Images in Clinical Medicine, a weekly Journalfeature, presents clinically important visual images, emphasizing those a doctor might encounter in an average day at the office, the emergency department, or the hospital. If you have an original unpublished, high-quality color or black-and-white photograph representing such a typical image that you would like considered for publication, send it with a descriptive legend to Kim Eagle, M.D., University of Michigan Medical Center, Division of Cardiology, 391 Taubman Center, Box 366, 15 East Medical Center Drive, Ann Arbor, MI For details about the size and labeling of the photographs, the requirements for the legend, and authorship, please contact Dr. Eagle at (phone) or (fax), or the New England Journal of Medicine at images@edit.nejm.org ( ).